1-(alkylsubstituted phenyl)imidazoles useful in acth reserve assay

ABSTRACT

DESCRIBED AND CLAIMED ARE THE 1-(ALKYLSUBSTITUTEDPHENYL) IMIDAZOLES OF THE FORMULA   1-(2-R1,3-R2,4-X,5-R3,6-R4-PHENYL)IMIDAZOLE   WHEREIN AT LEAST ONE OF R1 THROUGH R4 IS A SATURATED LOWER ALIPHATIC GROUP OF UP TO SIX CARBON ATOMS, THE REMAINING R GROUPS BEING HYDROGEN, AND X IS HYDROGEN WITH THE PROVISO THAT X CAN BE LOWER ALKYL WHEN BOTH R1 AND R4 ARE LOWER ALKYL. THE COMPOUNDS OF THE INVENTION POSSESS SELECTIVE ACTIVITY AS INHIBITORS OF STEROID HYDROXYLATION IN THE ADRENAL CORTEX AND ARE THUS USEFUL IN THE ACTH RESERVE ASSAY.

United States PatentfOifice 3,637,731 5 I l-(ALKYLSUBSTITUTED PHENYL)IMIDAZOLES USEFUL IN ACTH RESERVE ASSAY Alexander Lawrence Johnson, Wilmington, Del., assignor to E. I. du Pont de Nemours and Company, Wilmington, Del. No Drawing. Filed July 18, 1968, Ser. No. 745,672 Int. Cl. C07d 49/36 US. Cl. 260-309 7 Claims ABSTRACT OF THE DISCLOSURE Described and claimed are the l-(alkylsubstitutedphenyl) imidazoles of the formula BACKGROUND OF THE INVENTION (1) Field of the invention This invention relates to a novel group of l-(alkylsubstituted phenyl) imidazoles possessing selective activity in warm-blooded animals as inhibitors of adrenocortical steroid hydroxylation.

The anterior pituitary gland secretes important hormones, one of which is the adrenocorticotrophic factor designated ACIlH. This polypeptide of 39 amino acid residues, the structure of which was established by Bell and coworkers, acts upon the adrenal cortex stimulating the production of the adrenocortical hormones from the precursor progesterone. In this regard ACTH has been shown to mediate hydroxylation at the 115-, 18- and 2l-positions of the steroid nucleus.

Diagnostic aids to determine adrenocorticotrophic ability have been sought for some time. It has long been known that the regular sequence of enzymatic hydroxylation reactions in the adrenal cortex is apparently blocked in certain endocrine diseases. The result is that certain factors fail to be elaborated while their precursors accumulate. But it is only more recently that the administration of certain non-steroidal agents has been found to inhibit adrenocortical steroid hydroxylations. Among these compounds are amphenone B 3,3-bis-(p-aminophenyl)-2-butanone dihydrochloride] and SU-4485 also known as Metopirone [2-methyl-1,2-bis-(3-pyridyl)-1- propanone]. The latter compound appears to inhibit 11,9- hydroxylation specifically so that, when administered, the adrenal glands secrete ll-deoxycortisol instead of cortisol itself. The resumption of cortisol production after administration of SU-4485 is discontinued has suggested the use of this compound in testing pituitary ACTH reserve.

There is, however, a particular need for compounds useful in testing pituitary function that are less toxic than the compounds currently available. Such materials would be useful not only for regulation of corticoid dosage but also possibly'for the control of hypertension and electrolyte balance.

Accordingly, it has been found that the novel imidazoles of this invention are good diagnostic aids for the ACTH reserve assay, i.e. for testing pituitary function, in that they inhibit adrenocortical steroid hydroxylation in warm-blooded animals.

(2) Description of the prior art Several l-(substituted aromatic) imidazoles bearing a rather close structural relationship to the compounds of the present invention were synthesized by A. Wohl and W. Marckwald late in the nineteenth century. Preparation of l-(phenyl) imidazole was reported by these workers at Ber. 22, 568 (1889) and Ber. 22, 1353 (1889). Preparation of 1-(4-methylphenyl) imidazole, 1-(2,4-dimethy1- phenyl) imidazole and l-(l-naphthyl) imidazole was disclosed at Ber. 25, 2354 (1892). None of these compounds has been reported to act as an inhibitor of steroid hy droxylation in the adrenal cortex or to be useful in the ACTH reserve assay.

F. W. Kahnt and R. Neher in Helv. Chim. Acta 49, 725 (1966) describe the steroid hydroxylation blocking activity at the 1118, 17m, 18- and l9-positions of the steroid nucleus of 1-, 2- and 4(5)-(benzyl) imidazoles. Unlike the compounds of the present invention in which the aromatic function is bonded directly to imidazole nitrogen, thecompounds of Kahnt and Neher all possess a methylene link between imidazole and phenyl (i.e. they are of the benzyl type). Furthermore only in the case of l-benzyl imidazole is there imidazole substitution on ring nitrogen.

SUMMARY OF DETAILS OF THE INVENTION The new compounds of this invention are l-(alkylsubstituted phenyl) imidazoles having the structure wherein at least one of R through R is a saturated lower aliphatic group of up to six carbon atoms, the remaining R groups being hydrogen, and X is hydrogen with the proviso that X can be lower alkyl. The particularly preferred compounds of this invention which possess a high degree of inhibition of adrenocorticol steroid hydroxylation are those in which the groups R R and X of Formula I are hydrogen and the groups R and R of Formula I are aliphatic or cycloaliphatic groups which taken together have a total of 2 to 8 carbon atoms or which taken individually have up to 6 carbon atoms each. Also quite active and useful are those compounds in which the groups R and R whatever they may be, are substituted with aliphatic or cycloaliphatic groups as previously described. In the most active compounds of this invention either or both of R and R possess at least 3 carbon atoms.

l-(alkylsubstituted phenyl) imidazoles (I) may be pre pared by reacting an alkylphenyl isothiocyanate (III) with an aminoacetal (IV) to give an alkylphenyl acetalylthiourea (V) which undergoes acidcatalyzedring closure to produce the 2-rnercaptoimidazole having-alkylsubstituted phenyl in the 1-position (VI). The mercapto group is readily removed from the lattercornpound by treatment with an oxidizing agent to give the l-(alkylsubstituted phenyl) imidazoles of this invention (I). The process is illustrated by the following scheme:

4 two hours longer. The aqueous mixture was extracted three times with 50 ml. of chloroform, and the isothiocyanate (III) was isolated from the dried extract by distillation.

A solution of 30.0 g. (0.225 mole) of aminoacetal (IV) in 250 ml. of ethanol was treated with 0.225 mole of the isothiocyanate (III). After the addition was complete, the mixture was heated under reflux for 30 minutes and then stripped of ethanol. In a few cases, the intermediate aryl acetalylthiourea (V) was purified, but more usually it was converted directly the mercapto compound (VI) by heating for 30-60 minutes at reflux with 250 ml. of 10% aqueous hydrochloric acid. The mercaptans were isolated by cooling the acid solution to 0 and filtering the crystalline product. Purification was effected by recrystallization.

LA slurry of the Z-mercaptoimidazole (VI) and four times its weight of aqueous nitric acid was warmed on a steam bath in a large Erlenmeyer flask. After the vigorous reaction was over, the mixture was cooled, made basic with 15% aqueous ammonia and extracted with three portions of chloroform. The chloroform extracts were dried and evaporated to leave a residue of the crude imidazole which was purified by short-path distillation or by sublimation to give the l-(alkylsubstituted phenyl) imidazole (I).

Tables I and II which follow list the various alkyl-substituted phenyl isothiocyanates of Formula III and the l-(al- R- R kylsubstituted phenyl)-2-mercaptoimidazoles of Formula VI which were prepared as intermediates in the overall R3 R synthesis of the l-(alkylsubstituted phenyl) imidazoles of i Examples 1-11. All synthetic steps were carried out accord- X ing to the general experimental scheme previously detailed. (I)

TABLE I.ALKYLSUBSTITUTED PHENYL ISOTHIOCYANATES (111 B P. in C Yield R7 R3 R4 X (mm. Hg) percent 0H H H H 00 0.1 37 11 H OH; H 07 (0.4) 30 H H H H 32 1.3) 03 H H H H 53 0.1 70 H H CzHs H 82 (0. 88 011. H H H 50 0.1 75 H H H 11 00 0.1 32 8 3 H H: H II 0 (oHmCH H H cHmcH H 34 0.2) 34 10 CH2CH2CH2CH2 H H H 75 (0.05) 50 11 CH: H CH3 CH: 75 0.25 37 TABLE II.1-(ALKYLSUBSTITUTED PHENYDu-MERCAPIOIMIDAZOLES v1 Analysis (percent) Rocrystal- UV mu. Calculated Found lization M1./ Percent M1. N0. solvent g. yield C.) Solvent 011. 11131. C H N C H N o 1 021215011 20 203205 02115011 205 12,300 04.00 5.02 13.72 2 041150111 72 303-3045 041315011 204 14,000 04.00 5.02 13.72 3 05115011 0 03 225-2205 0411.011 205 11,700 00.03 0.47 12.34 4 04115011 4 37 200201 02115011 204 11,300 04.00 5.02 13.72 CzH OII 0 57 103-105 01115011 205 13,000 07.22 0.04 12.00 0 04115011 13 32 147-140 02115013 231 7,4 63-15 14-73 2;;3 (Used directly for I) 1 .4 3 33% 04115011 15 73 243-245 CZH3OII 203 12,000 03.15 5.30 14.73 A l. 0 04115011 3 32 243-245 CZHSOH 205 13,000 00.74 7.02 10.35 10... CzH5OH 0 37 213-215 CzH OH 203 12,000 07.31 0.13 12.17 0' 11..... CzHaOH 20 33 272-274 04115011 204 14,200 00.03 0.47 12.34 1 Extraction.

1 Decomposition.

The experimental details involved in preparation of the l-(alkylsubstituted phenyl) imidazoles of this invention according to the foregoing synthetic scheme are as follows:

To a rapidly stirred mixture of 24.9 g. (0.216 mole) of thiophosgene and 350 ml. of water there was added slowly 0.20 mole of the appropriately substituted aniline (II).

EMBODIMENTS OF THE INVENTION There follows in Table III a set of examples illustrative of the present invention. All compounds comprising the entries in this table were prepared through the intermediates enumerated in Tables I and II according to the gen- After the addition was complete, stirring was continued for eral experimental scheme previously described.

TABLE rrr-r-(ALxYLsoBs'rr ro'rnorfinrnin) IMIIEQAZOLES (I) Analysis (percent) Found Calculated max.

Yield Solvent Example l 5.. L 1-(2,6-diethylphenyl) imidazole 62 C2H5OH 9 1-(2,6-diisopropylphenyl) imidazole reserve assay. For example, when the process of the preceding examples is repeated using o-(2-methylbutyl) aniline, 2,4,6tri-tert-pentylaniline, o-hexylaniline (from aniline and hexene-l with Al/ClCl at 290 and 240 atm.),

4-indanamine, o-cyclopropylaniline, o-cyclopentylaniline, and o-cycl0hexylaniline, the compounds of this invention produced are 1 (2 [2 methylbutyl] phenyl) imidazole, 1 (2,4,6 tri tert pentylphenyl) imidazole, 1 (2 hexylphenyl) imidazole, 1 (4 indanyl) imidazole, 1 (2 cyclopropylphenyl) imidazole, 1 (2 cyclohexylphenyl) imidazole.

All of the compounds of this invention are in vitro and in vivo inhibitors of steroid hydroxylases, particularly the 18- and 2l-hydroxylases. They are useful in diagnosis and evaluation of the functional status of the hypothalmic-anterior pituitary. The following procedure illustrates the method by which the activity of the hydroxylation inhibitors of this invention may be demonstrated.

EXAMPLE A (1) Enzyme preparation Bovine adrenal glands were collected as soon as possible after slaughter, freed from connective tissues and adhering fat and placed in Dry Ice. The frozen glands were stored at 20 and processed Within 48 hours.

The following operations were carried out in a cold room at 4. The adrenal cortex of the glands was removed while still frozen and was homegenized with three times its weight of ice-cold 0.25 M sucrose solution, first in a Waring Blendor at speed for two minutes and then in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 700 g for 15 minutes to obtain the mitochrondrial fraction. This step was followed by centrifugation of the supernatant fluid at 105,000 g for 60 minutes to sediment the microsomes.

The mitochondria and the microsomes were washed by resuspension of these. fractions in twice their weight of 0.25 M sucrose solution and centrifugation at 700 g and 105,000 g for 45 minutes respectively.

The mitochondrial and microsomal pellets were resuspended in three times their weight of distilled water and lypophilized. The freeze-dried preparation was stored at 20 under desiccation.

The mitochondrial preparation was used for studying the llfl-hydroxylation of ll-deoxycorticosterone, the microsomal fraction for C-21 hydroxylation of progesterone and pregnenolone in vitro.

(2) Incubations the beakers in 0.05 ml. of propane- LZ-diol before other additions.

After incubation the steriods were extracted with 45 ml. of methylene chloride and centrifuged at 0 for 10 minutes, and the lower layer was quantitatively transferred to another tube-and evaporated under nitrogen. The extracted steroids were dissolved in a definite volume of methylene chloride and suitable aliquots were taken for the estimation of products.

(3) Assay methods To determine'the effect of'the compounds tested on steroid llfi-hydroxylation, ll-deoxycorticosterone was employed as the substrate. The product, corticosterone, was estimated fiuorometrically (Moncloa et al., Endostandard Metopirone the hydroxylation blocking activity of several prior art compounds.

TABLE V Activity (Metopi- Compound Reference rone"=1) A. Wohl and W. Marckwald Ber. 22 568 1,353 (1889) 0.05 W. Marckwald B01125 2,354 (1802) 0.

(EH3 W. Mnrckwald Ber. 25 2,354 (1802) 2 N\/N- orr W. Marckwald Ber. 25 2,354 (1892) 2 L F. Kahnt and R. Neher Helv. Chim.Acta 1,9 725 (1766) crinology, 65, 717 [1959]) as described below:

Aliquots of the sample corresponding to about 0.2, 0.4, 0.6, 1.0 and 2.5 g. of corticosterone were taken in thick-wall glass stoppered tubes With a capacity of -11 ml. The solvent was blown off under a stream of nitrogen, and the residue was dissolved in 2 ml. of 13% ethanol. Each sample was washed with 5 ml. of petroleum other by shaking for one minute, centrifuging for five minutes and removing the upper layer by aspiration. The sample was next extracted with 4 ml. of methylene chloride, and the aqueous phase was removed by aspiration. To the methylene chloride extract was added 1 ml. of icecold 0.1 N sodium hydroxide solution; the tube was shaken for 15 seconds, centrifuged for 5 minutes, and the sodium hydroxide layer was carefully removed. Three ml. of sulfuric acid reagent was then added; the tube was then shaken vigorously for 1 minute and finally centrifuged for 5 minutes. The sulfuric acid phase was removed and its fluorescence was determined after 50 minutes in an Aminco Bowman spectrophotofluor'ometer. For corticosterone the monochromator for activation wave length was set at 470 mi and that for recording the fluorescence at 515 my. With each set of observations, a five point curve for six samples (0.3 to 2.4 g.) of corticosterone was established as the standard.

Table IV compares the activity of the commercially available steroid hydroxylation inhibitor SU-4885 (Metopirone) with that of the l-alkylsubstitutedphenyl imidazoles of this invention. The Metopirone has been given an activity of unity in the procedure detailed above.

TABLE IV Table V presents for comparison with the commercial Since obvious modifications and equivalents in the invention will be evident to those skilled in the chemical arts, I propose to be bound solely by the appended claims.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. 1-(alkylsubstitutedphenyl)imidazole of the formula wherein X=R =H; and the substituents R R and R are selected from the groupings: R =isopropyl and R =H; R is ethyl and R -=R =H; R R =ethyl and R =H; R is tertiary butyl and R =R =H; 'R =R =is0propyl and R =H; and R and R together are CH -CH CH CH and R =H.

2. A compound of claim 1 wherein R =isopropyl and X=R =R =R =hydrogen, 1 (2 isopropylphenyl) imidazole.

3. A compound of claim 1 wherein R =ethyl and X=R R =R =hydrogen, 1-(2-ethy1phenyl)imidazole.

4. A compound of claim 1 wherein.R =-R =ethyl and X:R =R =hydrOgen, 1 (2,6-dietbylphenyl)imidazole.

5. A compound of claim 1 wherein R =t-butyl and X=R =R =R :hydrogen, 1 (2-t-butylphenyl)irnidazole.

6. A compound of claim 1 wherein R =R =isopropyl and X=R =R =hydrogen, l-(2,6-diisopropylphenyl) imidazole.

7. A compound of claim 1 wherein R and R together are CH CH --CH CH and X=R '=R*=hydrogen, 1-[8-(1,2,3,4-tetrahydronaphthyD]imiduzole.

References Cited Wohl et a1. II: Berichte, v01. 22, pages 1353-62 (1889) 10 10 Wohl et a1. III: Berichte, v01. 25, pages 2354-73 (1892) QDl.D4.

Kahnt et a1.: Helv. Chim. Acta, v01. 49, pages 725-32 (1966) QD1.H4.

5 NATALIE TROUSOF, Primary Examiner U.S. C1. X-R. 260999 

